The chemical profiles and cytotoxicity of gaharu bouya oil from Borneo’s Gonystylus bancanus wood

Gaharu bouya oil obtained from distillation of the woods from Gonystylus genus has attracted essential oil industry interest. However, the information about gaharu bouya essential oil profile is limited. The presence of Gonystylus species is also critically endangered on the IUCN Red List. Therefore, exploring the -omics profiles of Gonystylus bancanus, a native plant from Borneo Island, is important for Indonesia to conserve the population. This research investigated the metabolite profiling of G. bancanus oil, especially the volatile components of its essential oils. Distillations were performed in two technical ways: hydrodistillation on a laboratory scale and steam distillation on an industrial scale. According to LC–MS and GC–MS profiles, both essential oils displayed similar chemical compositions. This article also discusses the similarity of the chemical contents of gaharu bouya oil and agarwood oil from the gaharu superior type (Aquilaria) to support the value of the oil. This research also investigated the cytotoxicity of gaharu bouya oil against three cell lines: HeLa, MCF-7, and HT-29.


Discussion
Ramin wood, identified as Gonystylus bancanus, is a kind of hardwood from the Thymelaceae family.All parts of the wood (including sapwood and heartwood), except the bark, were processed together as the sample of G. bancanus wood.This wood has been determined for its taxonomy using species barcoding from the DNA sequencing method, and the result is presented in Fig. 1.The BLAST on NCBI results in two hits of related target DNA sequences, i.e., Gonystylus bancanus chloroplast (partial genome) (https:// www.ncbi.nlm.nih.gov/ nucco re/ EU849 490.1) and ribulose-1,5-biphosphate carboxylase/oxygenase large subunit (rbcL) gene (https:// www.ncbi.nlm.nih.gov/ nucco re/ KU244 237.1) with the percentage identities of 92.21% and 92.04%, respectively.The wood was also identified based on its wood anatomy with three slice forms of ramin wood in transversal, radial, and tangential sections, as seen in Fig. 2. The observation under the microscope on a slice of ramin wood showed that the wood grain (texture) had a straight direction and a smooth surface, with an oval-shaped porosity, and were mostly solitary cells.The parenchyma cells were axially paratracheal, with a thin wing, aliform, and some of them were short tangential bands, but no axial intercellular duct was found.It was assumed that gum conduction did not occur in this wood.This wood has a density of 0.60 g/cm 3 , which therefore was grouped in the wood with a strength of class III.A cross-section of the wood showed that the dark lines across this transverse section represented the cut of axial cells.A few dark wavy lines in the tangential longitudinal indicated the presence of latewood.Horizontal lines in the radial area of the wood represented the rays, while the vertical lines supported the fact that it was latewood, marked with densely-layered cells.
Microstructures of wood powder before and after the treatment were also observed using FESEM.Before the treatment, ramin wood powder appeared rough and hard, while after the treatment, the powder had less surface roughness, slightly smoother, and softer (Fig. 3E,F).The treatment created holes (Fig. 3B,D) due to the interaction of materials with hot water during distillation.These empty spaces were proposed to be formed mainly due to the loss of some chemical components or complex molecules extruding through the ramin wood surfaces.Physically, the treatment affected the topology, roughness, and chemical composition of materials, as shown by the images of the epidermic layer on the wood surfaces between the left and right sides in Fig. 3.
Distillation is a standard method for extracting essential oil from plant parts.Gaharu bouya oil produced by hydrodistillation resulted in a 0.90 ± 0.01% yield, while industrial scale using steam distillation resulted in an oil Chromatography analysis of gaharu bouya oils was performed using GCMS and LCMS (Fig. 4).Chromatograms from GCMS data showed that both gaharu bouya oils exhibited a similar pattern of peaks for the compounds represented inside the oils.Both oils contained two significant peaks, which were not significantly different, but both mass spectral data predicted different compounds in detail.Two major peaks in GCMS data from commercial gaharu bouya oil represented 10-epi-γ-eudesmol and β-eudesmol, while from this research, gaharu bouya oil signified 10-epi-γ-eudesmol and α-eudesmol.However, LCMS data strengthen the results that eudesmol isomers, i.e., 10-epi-γ-eudesmol, β-eudesmol, and α-eudesmol, are similar in the significant amounts of the oils.The predicted compound, γ-eudesmol, also presented in the commercial gaharu bouya oil as an isomer of 10-epi-γ-eudesmol, hence the amounts of 10-epi-γ-eudesmol would be the total of both isomers of γ-eudesmol.The Venn diagram of chemical components in both essential oils from two distillation methods in Fig. 5 also shows an intersection as an overlap of 38 components in both oils, including two eudesmol isomers, i.e., β-eudesmol and 10-epi-γ-eudesmol.In the other hand, there are 34 components exclusively found in essential oil from steam distillation, and 21 other compounds are in hydrodistillation product.
Figure 7 shows that the analysis of LCMS and GCMS data (from Table 1) can be remarkably distinguished by PC1 (F1).The LCMS data resulted in various chemical components in the superior quadrant, while the GCMS data was in the negative quadrant of PC1.Interestingly, LCMS data of both samples (HD and SD) also resumed high variability of components separated by PC2 (F2).However, GCMS data of gaharu bouya oil from this research and the commercial one showed high similarity in chemical constituents.This PCA biplot implied that volatile compounds detected by GCMS analysis were similar in both samples (Table 1).It was also concluded that alcoholic sesquiterpenes with eudesmane skeleton, especially 10-epi-γ-eudesmol, β-eudesmol and α-eudesmol, which were the major volatile compounds, took more than 80% of oil compositions in gaharu bouya oil (Table 3).
According to that, although both distillation methods share 38 common components in their essential oils, including 10-epi-g-eudesmol, but the total components responsible for the fragrant odor, particularly for terpenoids (the sesquiterpenes) and benzene derivatives, are shown to be higher in the steam distillation product than in hydrodistillation.The physical performance of the essential oil from steam distillation appears transparent,  www.nature.com/scientificreports/viscous, and no solid form at fridge temperature (4 °C).In contrast, the oil from hydrodistillation forms a white solid lipid at low temperatures, which liquefies when the temperature rises.The oil yields obtained from both distillations showed that steam distillation produced a 10% higher oil yield than hydrodistillation.Therefore, it is suggested that steam distillation is a preferred method to be applied on the industrial scale of essential    www.nature.com/scientificreports/oil production since it produces higher oil yield, better physical performance, and metabolically satisfactory eudesmol content than hydrodistillation.β-eudesmol, another eudesmol available in gaharu bouya, has been known to show a wide range of bioactivities in previous research 20 .Antimutagenic activity by suppressing umu gene expression 48% has been reached by applying β-eudesmol at less than 0.18 μmol/mL 21 .10-100 μM of β-eudesmol inhibited proliferation of HeLa, SGC-7901, and BEL-7402 22 .It is also potential as an anti-cholangiocarcinoma candidate with IC 50 value of 47.62 ± 9.54 μM at 24 h interaction against KKU-100 cells 23 .It inhibited the growth of HL-60 cells with an IC 50 value of 35.1 μM 24 and induced DNA fragmentation in HL-60 cells at 80 μM 25 .Remarkably, 950 ng of β-eudesmol intake could affect mental stress in humans 26 .
According to those references, cytotoxicity assays against some cell lines were carried out using gaharu bouya oil containing some eudesmol derivatives: β-eudesmol, 10-epi-γ-eudesmol, and α-eudesmol.Three cell lines, including HeLa, MCF7, and HT-29 have been used to represent cervical cancer, breast cancer, and human colon cancer cells, respectively (Fig. 6).Unfortunately, the cytotoxicity assay results for G.bancanus wood essential oils against those three cell lines were not in accordance with other research results on bioassay of β-eudesmol [22][23][24][25] .
The IC 50 values of gaharu bouya oils took about 10-400 times more than cisplatin, the standard chemotherapy medication (Table 2).It showed that only the MCF-7 cell line was inhibited in a similar concentration to cisplatin.However, the relatively high value reaches more than 100 μg/mL.Consequently, using gaharu bouya oil as a chemotherapeutic agent for several diseases including cervix, breast, and human colon cancer is impossible.However, the isolation of β-eudesmol and two other eudesmols: 10-epi-γ-eudesmol and α-eudesmol, as three major volatile compounds in gaharu bouya oil would be an essential step to conduct, followed by the analysis of its bioactivities against some cancer cell lines stated before.

Conclusion
Gaharu bouya oil from Gonystylus bancanus contained mostly volatile components from terpenoids and benzene aromatics groups.The highest proportion component of gaharu bouya oil is 10-epi-γ-eudesmol, a marker compound found in gaharu superior from Aquilaria malaccensis.However, steam ditillation gains more advantageous for being applied in industrial scale since it produces higher oil yield, better oil physical performance, and comprehensive metabolomic than hydrodistillation.This fact broadens the benefit of gaharu bouya oil production from ramin wood since it can be beneficial as an additive or substitute for agarwood oil.Although cytotoxicity assays of gaharu bouya oil against HeLa and HT-29 did not positively impact inhibition, they showed comparable activity to cisplatin as the standard against MCF-7 cells.Therefore, gaharu bouya oil has a potency to be explored further, especially in isolating its chemical components, and to be investigated for its bioactivity as a candidate for an anti-breast cancer agent.

Plant materials
The wood of Gonystylus bancanus (ramin) was initially obtained from the Kalimantan, Indonesia forest.This wood is not taken from its natural habitat by logging but from the wood parts that have fallen.Ramin wood has been marketed into Java as a legal log.The current sample of this research was obtained from the manufacturer and exporter of essential oils, PT Padaelo Sejahtera, Magelang, Central Java, Indonesia, which distributed the wood chips legitimately.This ramin wood (Fig. 9) was identified by species barcoding method from PT. Genetika Science Indonesia, and by wood anatomists Prof. Agus Sulistiyo Budi and Sri Wahyuni.This wood was acknowledged as a part of Gonystylus bancanus, and was deposited at the Laboratory of Biology and Wood Preservation, Faculty of Forestry, Universitas Mulawarman, Indonesia, under voucher number 220622-3.The supporting data for this identification was presented in the results and discussion section.The wood chips of ramin were ground into powder using a miller processor.This powder was subjected to further distillation to produce gaharu bouya oil.The commercial gaharu bouya oil was obtained from steam distillation of the same ramin wood provided by PT.Padaelo Sejahtera.The experimental field study adheres to pertinent institutional policies and conforms with pertinent laws.

Methods
The methodology of this research consisted of six parts: (1) species barcoding of wood material; (2) characterization of wood chip materials; (3) isolation of essential oil using hydrodistillation; (4) total phenolic and flavonoid content; (5) chromatography analysis for chemical components identification, including LCMS and GCMS; (6) statistical analysis based on Principal Component Analysis (PCA); (7) cytotoxicity assays against three cell lines (HeLa, MCF7, and HT-29).

Species barcoding of wood materials
The wood sample was extracted for its DNA with Quick-DNA Plant/Seed Miniprep Kit (Zymo Research, D6020).PCR amplification was carried out using My Taq HS Red Mix kit, 2X (Bioline, BIO-25048).The electrophoresis of the amplified product was conducted on agarose gel 1% in TBE buffer based on rbcL gen.Bi-directional sequencing was applied based on Sanger DNA Sequencing by using Capillary Electrophoresis to produce a bioinformatic analysis result related to BLAST against NCBI database.

Characterization of wood materials
Characterization of wood materials as samples in this research included surface analysis of the slicing wood using a light microscope (Olympus BH2) and microstructure analysis of wood powder using FESEM Hitachi type Regulus 8220.FESEM (Field Emission Scanning Electron Microscopy) sample imaging was obtained from samples prepared as gold-coated specimens and imaged under high vacuum at 1.0 kV and 1.5 kV.SEM images were recorded at magnifications ranging from × 250 to × 11.00.

Distillation of ramin wood
Five hundred grams of wood powder was distilled using a Clevenger hydrodistillation set-up with an addition of 3.5 L aquadest.Distillation was run for 6 h and was counted for the first second of distillation at the first drop of distillate.The essential oil in the distillate was separated from the water and was dried using anhydrous magnesium sulfate.Resinous oil was obtained and kept at 5 °C until it was sent for further analysis using GCMS, LCMS, and bioassay.Another sample of essential oil used in this research was obtained privately from an essential oil distiller, PT.Padaelo Sejahtera, with purity of 100% without any solvent addition.This gaharu bouya oil was extracted on an industrial scale using steam distillation for 36 h.Further analysis of this oil was delivered using LCMS and GCMS.

Total phenolic and flavonoid contents
Total phenolic content was determined based on the regular method applied in our laboratory 27,28 .The Modified Folin-Ciocalteu method was employed by mixing the sample (1000 ppm) with 10% Folin-Ciocalteu solution and 5% sodium carbonate.The total phenolic content was measured as absorbance at 765 nm to compare with gallic acid, as standard, using spectrophotometer Genesys, Thermo Fisher Scientific, Madison, WI, USA.This number was also expressed as a mg GAE (gallic acid equivalent) per gram of essential oil.
Total flavonoid content was evaluated using the modified Aluminium Chloride method as reported in the previous articles 27,29 .Briefly, the sample was mixed with 2% AlCl 3 in methanol and incubated for 1 h at room temperature.The mixture was measured at 415 nm using a UV-Vis spectrophotometer.The absorbance of the sample was compared to a standard curve of quercetin.Total flavonoid content was calculated as mg QE (quercetin equivalent) per gram of essential oil.

Chromatography analysis
Liquid Chromatography Mass Spectrometry (LCMS) analysis was performed on a Shimadzu LCMS-8040 LC/ MS equipped with a Shimadzu Shim Pack FC-ODS column of 2 mm × 150 mm, and 3 µm with mobile phase mode was isocratic with a flow rate of 0.5 mL/min and a sampling cone of 23.0 V.The MS-focused ion mode was ion type [M] + with a collision energy of 5.0 V, desolvation gas flow of 60 mL/h, and desolvation temperature of 350 °C.The fragmentation method was low energy CID with ionization by ESI, scanning rate was 0.6 s/scan (m/z: 10-1500), source temperature was 100 °C, and run time was 80 min.
Gas chromatography-mass spectrometry (GCMS) analysis was run on Shimadzu GCMS-QP2010S equipped with DB-5MS column in 30 m length, 0.25 mm diameter, and 0.25 μm wide of a film with the column oven temperature was 70 °C held in 5 min, while injection temperature was 300 °C for 19 min.The ion source temperature for MS was 250 °C, the interface temperature was 305 °C with a solvent cut time of 3 min, and the detector gain mode was relatively + 0.00 kV.
Spectrums and their fragmentations obtained from LCMS and GCMS analysis were matched to the spectrum references under Mass Spectral Library of NIST20 and WILEY229-NIST62 databases, respectively.The instruments are regularly standardized using a reference mass of perfluorotributylamine (PFTBA, C 12 F 27 N) and PEG-PPG-Raffinose, respectively.These databases confirm a range of volatile and non-volatile compounds.

Statistical analysis
The diagnostic tool for statistical analysis in this research included a score plot and loading plot (shown in the biplot figures) of Principal Component Analysis (PCA), a dendrogram of Hierarchical Clustering Analysis (HCA), and a 3D plot of Origin software.A biplot analysis was performed on data from LCMS and GCMS analysis as a graph of extraction solvents toward the relative amount of correlated identified compounds.The clustering of the extracts was determined from the identified compounds resulting from the variation of solvent extraction and was mapped on HCA.Whenever the accumulative eigenvalue of PCs was less than 80.0%, an increasing matrix was compulsory to apply until the minimum PC value of 80.0% was obtained.

Cytotoxicity assay
Anticancer properties were identified by testing gaharu bouya oils against three cell lines: HeLa, MCF7, and HT-29.Cytotoxicity assay was performed based on the MTT method using CellQuanti-MTT™ Cell Viability Assay Kit (Cat.No. CQMT-500).The protocol is available on the BioAssay Systems website 30 .Anti-proliferative effects of those cell lines were observed using an inverted microscope and Elisa reader.

Figure 1 .
Figure 1.Sequence assembly (586 bp) result from the amplified product and the top two hit BLAST related to DNA target of Gonystylus bancanus.

Figure 2 .
Figure 2. The illustration of ramin wood as Gonystylus bancanus is based on three sections: transverse (crosssection), radial, and tangential sections, observed under a light microscope.

Figure 5 .
Figure 5. Venn diagram of identified compounds in the essential oils from steam distillation and hydrodistillation method obtained from the data of LCMS and GCMS at Table 1.

Figure 9 .
Figure 9.The ramin wood as part of Gonystylus bancanus.

Table 1 .
Chemical components in gaharu bouya oils analyzed by LCMS and GCMS.SD steam distillation, HD hydro distillation.

Table 2 .
Comparison of G. bancanus oil and positive control (cisplatin)'s concentrations at fifty percent inhibition to the culture cells: Hela, MCF7, and HT-29.

Table 3 .
Major components in gaharu bouya oil based on LCMS and GCMS analysis.